The Center for Disease Control and Prevention has named Coxiella burnetti, the causative agent of the highly debilitating illness Q-fever, a Class B biothreat agent. Because of the difficulty in growing this organism, requirement for specialized containment facilities, and the lack of molecular genetic tools to manipulate its genome, very little progress has been made in understanding the molecular mechanisms of C. burnetti virulence and immune recognition. C. burnetti is an obligate intracellular pathogen that replicates within endoysosomal compartment that is actively modified by the bacterium, presumably by secreting "effector" proteins via a Type IV secretion system. We propose to identify Coxiella effector proteins by performing a functional genomic analysis of Coxiella proteins in a S. cerevisiae expression system. We have developed inexpensive high-throughput cloning methodologies to rapidly create a comprehensive, ordered array of yeast strains expressing a bacterial proteome as epitope-tagged protein fusions. These recombinant yeast strains will be screened to identify C. burnetti factors that 1) modulate conserved eukaryotic cellular functions, 2) are tropic for major eukaryotic organelles and 3) are exported from the parasitopherous vacuole. Candidate effector proteins will be validated in mammalian expression systems and with specific antibodies. The identification secreted virulence factors is the starting point for a broader understanding of the molecular basis of the pathogenesis of this organism and the design of vaccines candidates to boost cellular immunity. [unreadable] [unreadable] Health Relevance: The Center for Disease Control and Prevention has named Coxiella burnetii, the causative agent of Q-fever, a category B biothreat agent. Because C. burnetii is difficult to culture, requires specialized containment facilities and is not amenable to genetic analysis, very little is known about the molecular basis for its pathogenesis. In this grant application we propose experiments that take advantage of information available through genome sequencing efforts to identify and characterize Coxiella virulence factors. We predict that these factors will be attractive candidates for the design of new therapeutics and vaccines. [unreadable] [unreadable] [unreadable] [unreadable]